A runaway vector for mammalian cells was constructed from the simian virus 40 (SV40) genome with a temperature-sensitive mutation of the large T antigen and bacterial neor gene. Replication of this plasmid was repressed above 39°C and vigorous DNA propagation was observed below 33°C in simian CV-1 cells. The human erythropoietin gene was inserted downstream of the SV40 late promoter of the plasmid and the recombinant plasmid was introduced into CV-1 cells. By a temperature shift from 37 to 33°C, the plasmid copy number increased from 5 × 102 to 5 × 103 copies per cell and the specific production rate of erythropoietin increased more than ten-fold. The bacterial-derived sequences such as the neor gene and vector pUC sequences were prone to delete but the main body of the recombinant plasmid such as SV40 and the erythropoietin-coding sequences were stably maintained at either 33 or 37°C.
All Science Journal Classification (ASJC) codes
- Applied Microbiology and Biotechnology